The present invention relates to a G protein-coupled receptor which is expressed predominantly in lymphoid cells, is transcriptionally induced in response to proliferative stimuli and genotoxic treatment and integrates diverse cellular signals by modulation of cytoskeletal architecture.
The family of G protein-coupled receptors (GPCRs) has at least 250 members (Strader et al. FASEB J., 9:745-754, 1995; Strader et al. Annu. Rev. Biochem., 63:101-32, 1994). It has been estimated that one percent of human genes may encode GPCRs. GPCRs bind to a wide-variety of ligands ranging from photons, small biogenic amines (i.e., epinephrine and histamine), peptides (i.e., IL-8), to large glycoprotein hormones (i.e., parathyroid hormone). GPCRs play important roles in diverse cellular processes including cell proliferation and differentiation, leukocyte migration in response to inflammation, and cellular response to light, odorants, neurotransmitters and hormones (Strader et al., supra.).
Ligand binding to GPCRs elicits activation of signaling pathways via associated heterotrimeric G proteins comprising xcex1, xcex2 and xcex3 subunits. Heterotrimeric G proteins are classified according to the xcex1 subunit, which can be any of more than 20 grouped into 4 classes termed Gs, Gi, Gq and G12. Similarly, there are 6 known xcex2 and 12 xcex3 subunits, further increasing the complexity, although not all possible combinations are functional (Schmidt et al., J. Biol. Chem. 267:13807-13810, 1992). Initiation of signal transduction follows exchange of GTP for GDP bound to the xcex1-subunit and its dissociation from the xcex2xcex3 dimer. Both released GTP-bound xcex1 and free xcex2xcex3 components are capable of mediating signaling events through their interaction with effector molecules, leading to an appropriate physiological response. Importantly, activation of G protein signaling is integrated with receptor downregulation via serine/threonine phosphorylation of critical residues within the carboxy terminal cytoplasmic tail of the GPCR by GPCR kinases (GRKs) as well as Protein Kinase A (PKA) and Protein Kinase C (PKC) leading to desensitization and internalization of the receptor (Ferguson et al., Biochem. Soc. Trans. 24:953-959, 1996).
A growing number of GPCRs with ligand independent activity exist whose biological function is most likely regulated at the transcriptional level (Leurs et al., Trends Biochem. Sci. 23:418-422, 1998). While members of this class of GPCR are also subject to phosphorylation dependent downmodulation, transcriptional control of receptor number confers a high degree of temporal control over receptor turnover and activity.
Multiple GPCRs in a given cell-type can couple to the same G protein and a variety of accessory molecules exist which can modify both the responsiveness of GPCRs to effector signals (McLatchie et al., Nature 393:333-339, 1998) as well as integrating GTP exchange on Gxcex1 subunits with parallel signal transduction pathways to modify the biological outcome (Kehrl, Immunity 8:1-10, 1998; Strittmatter et al., Nature 344:836-841, 1990). Nevertheless, biological/biochemical responses to activation of a GPCR are determined primarily by the nature of the Gxcex1 subunits to which it is coupled, and this cannot be predicted by primary sequence analysis of newly discovered GPCRs (Hedin et al., Cell Signal 5:505-518, 1993). A primary objective, therefore, in the study of an orphan GPCR is to define its Gxcex1 coupling profile. While direct experimental approaches such as photolabelling of Gxcex1 subunits with radiolabeled GTP analogues have been useful in studies of GPCRs with known ligands or agonists (Offermanns et al., Meth. Enzymol. 195:286-301, 1991), they are limited in their application to the study of GPCRs in the absence of a defined ligand/agonist. However, signaling events downstream of many Gxcex1 subunits have been well defined and their analysis can serve as surrogate assays of Gxcex1 coupling profiles. Indeed, the biochemical/signaling properties of GPCRs are most often recapitulated in heterologous cell-types (Beadling et al., J. Immunol. 162:2677-2682, 1999) and cell lines in which the spectrum of expressed Gxcex1 subunits are defined can serve as systems in which to study the primary signal transduction and biological characteristics of orphan GPCRs. Such approaches have therefore been used for preliminary analyses of orphan GPCRs as well as for ligand/drug screening protocols (Fraser, J. Nucl. Med. 36:17S-21S, 1995).
Interest in this family of receptors has increased with the realization of their potential clinical applications as drug targets. From the perspective of their clinical significance there is considerable focus on GPCRs expressed in the hematopoietic and lymphoid systems as many have been shown to play pivotal roles in the regulation of hematopoiesis and immune function. Receptor/ligand relationships within the GPCR family exhibit significant promiscuity, with many receptors recognizing more than one ligand and vice versa. This is especially true among chemokine receptors and a major goal, therefore, is to define the spectrum of receptor/ligand interactions within this family of GPCRs, which includes a number of lymphoid expressed orphan receptors of unknown function.
Interestingly, GPCRs have functional homologues in human cytomegalovirus and herpesvirus, suggesting that GPCRs may have been acquired during evolution for viral pathogenesis (Strader et al., FASEB J., 9:745-754, 1995; Arvanitakis et al. Nature, 385:347-350, 1997; Murphy, Annu. Rev. Immunol. 12:593-633, 1994).
The importance of G protein-coupled receptors is further highlighted by the recent discoveries that its family members, chemokine receptors CXCR4/Fusin and CCR5, are co-receptors for T cell-tropic and macrophage-tropic HIV virus strains respectively (Alkhatib et al., Science, 272:1955, 1996; Choe et al., Cell, 85:1135, 1996; Deng et al., Nature, 381:661, 1996; Doranz et al., Cell, 85:1149, 1996; Dragic et al., Nature, 381:667 (1996); Feng et al., Science 272:872, 1996). It is conceivable that blocking these receptors may prevent infection by the human immunodeficiency (HIV) virus.
Cell cycle checkpoints, intervals in the cell cycle in which the cell detects impairment or loss of integrity to its genome and arrests growth in order to make repairs, ensure that DNA is replicated with high fidelity (Paulovich et al., Cell 88:315-321, 1997; Hartwell, Cell 71:543-546, 1992). There are three separately defined times in the eukaryotic cell cycle identified as checkpoints: G1/S transition, S-phase delay and G2/M transition (Nurse, Cell 91:865-867, 1997). The G1/S checkpoint is activated to avoid copying mutated DNA by increasing the time available for repair. Cells also utilize a DNA damage checkpoint within S phase by slowing the rate of DNA replication. The G2/M checkpoint is activated upon detection of double-stranded DNA breaks. In addition, mitotic entry is monitored by a spindle checkpoint that inhibits anaphase progression when chromosomes are not attached to the mitotic spindle (Nicklas, Science 275:632-637, 1997). The cell cycle checkpoint is summarized in FIG. 1.
Recent discoveries have shed light on the molecular participants in the G2/M transition. Cdc2 and Cyclin B1 promote entry into mitosis and are part of the maturation promoting factor (MPF). Dephosphorylation of Cdc2 on Thr14 and Tyr15 by Cdc25 and phosphorylation on Thr161 concomitant with nuclear association with Cyclin B1 results in rapid entry into mitosis. Cyclin B1 degradation or export to the cytoplasm and phosphorylation of Cdc2 on the negative regulatory sites Thr14 and Tyr15 by Wee1 block entry into mitosis. Caffeine can relieve DNA damage-activated G2/M arrest by stimulating the dephosphorylation of Cdc2. These data strongly implicate MPF as the central regulator of the transition from G2 into mitosis.
Recent work has broadened our understanding of the signaling pathways involved in G2/M arrest upstream of MPF. Response to DNA damage is detected by the Ataxia Telangiectasia mutated (ATM) which is a human homologue of the yeast rad family of genes (Meyn, 1995). The ATM protein has been implicated in the activation of Chk1, which phosphorylates Cdc25, leading to binding and sequestering of Cdc25 by 14-3-3 (Sanchez et al., Science 277:1497-1501, 1997; Peng et al., Science 277:1501-1505, 1997; Fumari, Science 277:1495-1497, 1997). This results in accumulation of the phosphorylated (inactive) form of Cdc2 and G2/M arrest. Cds1 has been demonstrated to function redundantly to Chk-1 by phosphorylating both Wee1 and Cdc25, inactivating both gene products (Boddy et al., Science 280:909-912, 1998; Fumari et al, supra.; Sanchez et al., supra.). ATM serves to activate proteins that act directly on MPF and lead to cell cycle arrest.
ATM also associates with and activates proteins that stimulate transcription of secondary molecules involved in checkpoint controls. One of these downstream activators of ATM is the tunor suppressor p53. Activation of p53 leads to the induction of multiple genes, including p21Cip and 14-3-3 (Levine, Cell 88:323-331, 1997). The 14-3-3 gene product mediates G2/M arrest by binding to Cdc25 to sequester it in the cytoplasm. The tyrosine kinase Abl physically interacts with the ATM gene product (Shafinan, Nature 5 387:520-523, 1997; Baskaran, Nature 387:516-519, 1997). Activation of the Abl kinase by DNA damage is dependent on the ATM, suggesting a functional link of Abl and ATM in the DNA damage checkpoint regulation. The overall regulation of the G2M checkpoint is an intricate mechanism involving both post-transcriptional modifications and transcriptional activation to guarantee proper cell growth. Thus, the known G2/M checkpoint proteins ultimately function through regulation of Cdc2 phosphorylation and nuclear import of Cyclin B1.
While the general eukaryotic cell cycle control machinery is highly conserved among a broad range of cell types, little is known about tissue-specific cell cycle regulators. TGF-xcex2 and GATA-5 represent anti-proliferative signaling molecules that are restricted in expression. Both of these regulators restrict the cell cycle at G1. Lymphocytes provide an interesting model system to study tissue-specific cell cycle regulators since their development is marked by the unique property of entering, exiting and re-joining the cell cycle depending on their internal developmental stages as well as the surrounding environment. For example, upon interaction with antigen, the resting mature naive B cells accumulate in the lymphoid germinal centers in which they undergo vigorous proliferation and excess B cells die by being included from germinal centers.
Loss of cellular growth controls by oncogenic transformation is dependent on signals emanating from the oncogene to downstream signaling partners and frequently leads to transcriptional induction of secondary genes which contribute to malignant growth. BCR-ABL is a chimeric tyrosine kinase oncogene generated by a reciprocal chromosomal translocation t(9;22)(q34;q11) associated with the pathogenesis of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL) (Kurzrock, N. Engl. J. Med. 319: 990-998, 1988). This chimeric oncogenesis found in Ph1-positive stem cells. Structural and functional analysis have defined critical domains within BCR-ABL responsible for its oncogenic activity. In particular, the R552L substitution within a highly conserved motif of the Src Homology 2 (SH2) domain uncouples the SH2 domain with phosphotyrosine-containing proteins without affecting the kinase activity of BCR-ABL. Interestingly, this mutation greatly reduces the ability of BCR-ABL to stimulate anchorage-independent growth of rat fibroblasts in soft agar (Goga, Cell 82:981-988, 1995). Although the SH2 mutant still retains the ability to transform primary bone marrow cells in vitro, it exhibits diminished malignant and leukemogenic potential in mice (Goga, supra.). Inactivation of the SH2 domain may uncouple BCR-ABL with downstream signaling molecules, which in turn may alter the expression of critical genes involved in leukemogenesis.
A method for identifying a compound which inhibits T cell hyperproliferation, comprising the steps of: contacting a G2A receptor with a test compound; determining whether the compound binds to said G2A; and if the compound binds to G2A, detemininig whether the compound activates said G2A receptor, whereby activation of the receptor indicates that the compound is a potential inhibitor of T cell proliferation. In one aspect of this preferred embodiment, the T cell hyperproliferation is associated with an autoimmune disorder, inflaummatory disorder or malignancy. Preferably, the T cell hyperproliferation is associated with a disorder selected from the group consisting of rheumatoid arthritis, psoriasis, inflammatory bowel disease, T cell an immature B cell malignancies and diabetes. In one aspect of this preferred embodiment, the G2A is expressed on the cell surface. Advantageously, the determining step comprises a high density bone marrow transformation assay.
The present invention also provides a method for inducing cell cycle arrest in a cell, comprising contacting said cell with a compound which activates the G2A receptor. Preferably, the cell cycle arrest occurs at the G2/M transition of the cell cycle. In one aspect of this preferred embodiment, the cell is a leukemia cell or lymphoma cell.
Another embodiment of the present invention is a method for determining the presence of cancer cells, comprising determining whether the cells express the G2A transcript, wherein the presence of an increased level of the transcript compared to a control cell indicates that said cell is a cancer cell. Preferably, the determining step comprises Northern hybridization or polymerase chain reaction.
The present invention also provides a method for detecting the presence of cancer cells, comprising determining whether the cells express G2A protein, wherein the presence of an increased level of the protein compared to a control cell indicates that the cell is a cancer cell. Preferably, the determining step comprises contacting the cells with an antibody specific for the G2A protein and detecting the presence of the antibody. In one aspect ofthis preferred embodiment, the detecting step comprises fluorescence activated cell sorting (FACS).